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Analysis of core region from egg white lysozyme forming amyloid fibrils.

Tokunaga Y, Sakakibara Y, Kamada Y, Watanabe K, Sugimoto Y - Int. J. Biol. Sci. (2013)

Bottom Line: The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C.A substantial number of fibrils were generated under this pH condition and incubation period.Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Bioscience The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065 Japan.

ABSTRACT
Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.

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Related in: MedlinePlus

Changes in ANS fluorescence spectra of the K peptide during incubation. The K peptide was incubated for 0 to 14 days at pH 4.0 at the concentration of 2 mg/ml as detailed in Materials and Methods and after dilution to 80 μg/ml, subjected to the analysis of ANS fluorescence emission. Excitation wavelength was set to 380 nm and emission wavelength to 420-540 nm.
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Figure 5: Changes in ANS fluorescence spectra of the K peptide during incubation. The K peptide was incubated for 0 to 14 days at pH 4.0 at the concentration of 2 mg/ml as detailed in Materials and Methods and after dilution to 80 μg/ml, subjected to the analysis of ANS fluorescence emission. Excitation wavelength was set to 380 nm and emission wavelength to 420-540 nm.

Mentions: Conformation alterations of the K peptide fibrils in terms of the exposed hydrophobic surface were investigated by the ANS binding assay. As illustrated in Fig. 5, extensive augmentation (approx 50%) of ANS fluorescence occurred as the incubation time of the K peptide proceeded. Moreover, there was a blue shift in the λ max from 515 nm (before incubation) to 490 nm (through incubation). This suggests that the formation of an exposed hydrophobic surface on the K peptide fibrils proceeded during incubation at pH 4.


Analysis of core region from egg white lysozyme forming amyloid fibrils.

Tokunaga Y, Sakakibara Y, Kamada Y, Watanabe K, Sugimoto Y - Int. J. Biol. Sci. (2013)

Changes in ANS fluorescence spectra of the K peptide during incubation. The K peptide was incubated for 0 to 14 days at pH 4.0 at the concentration of 2 mg/ml as detailed in Materials and Methods and after dilution to 80 μg/ml, subjected to the analysis of ANS fluorescence emission. Excitation wavelength was set to 380 nm and emission wavelength to 420-540 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3584918&req=5

Figure 5: Changes in ANS fluorescence spectra of the K peptide during incubation. The K peptide was incubated for 0 to 14 days at pH 4.0 at the concentration of 2 mg/ml as detailed in Materials and Methods and after dilution to 80 μg/ml, subjected to the analysis of ANS fluorescence emission. Excitation wavelength was set to 380 nm and emission wavelength to 420-540 nm.
Mentions: Conformation alterations of the K peptide fibrils in terms of the exposed hydrophobic surface were investigated by the ANS binding assay. As illustrated in Fig. 5, extensive augmentation (approx 50%) of ANS fluorescence occurred as the incubation time of the K peptide proceeded. Moreover, there was a blue shift in the λ max from 515 nm (before incubation) to 490 nm (through incubation). This suggests that the formation of an exposed hydrophobic surface on the K peptide fibrils proceeded during incubation at pH 4.

Bottom Line: The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C.A substantial number of fibrils were generated under this pH condition and incubation period.Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Bioscience The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065 Japan.

ABSTRACT
Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.

Show MeSH
Related in: MedlinePlus